The long-term goal of this proposal is to understand the role of intracellular calcium signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virus-infected cells and cancer cells and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. Understanding CTL function is therefore important for preventing and treating naturally occurring viral diseases such as AIDS and influenza, and viral diseases such as smallpox used as biological weapons. It is also important for understanding and treating cancers and autoimmune diseases. Finally, the ability to suppress CTL function is vital for successful organ transplantation. One of the main mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzymes contained in specialized lytic granules into an area of close apposition formed with the target. Granule exocytosis is known absolutely to require increased intracellular calcium caused by influx across the plasma membrane. However, the specific role(s) of calcium in granule exocytosis are unknown, the number of calcium-dependent steps is unclear, and molecules that confer calcium-dependence have not been identified. The specific aims of this proposal will use a battery of techniques, including novel fluorescence imaging methodologies we have developed, to: 1) determine whether bulk cytosolic calcium increases are sufficient to support granule exocytosis, or whether higher-than-cytosolic calcium increases in microdomains are required. 2) Investigate the calcium dependence of granule reorientation and of reorientation-independent exocytosis. 3) Determine whether immunological synapse formation is calcium dependent, and acts as a slow step in granule reorientation. 4) Investigate the role of the calcium-dependent phosphatase calcineurin in granule exocytosis. These studies will significantly further our understanding of the role of calcium influx in lytic granule exocytosis.